The present invention relates to novel collagenic peptides chemically modified by grafting free or substituted thiol functions, borne by mercaptoamino residues. When the collagenic peptides comprise thiol functions, they have the property of being crosslinkable by oxidation and give a collagen derivative crosslinked with disulfide bridges.
The invention is also directed toward a process for preparing these novel collagen derivatives which are in crosslinkable form, in the form of a crosslinkable precursor of a derivative or in crosslinked form.
The invention also relates to the uses of these novel collagenic peptides as biomaterials that are useful as starting materials for the manufacture of medical, surgical or cosmetic products, such as artificial tissues or organs, artificial skin, bone, ligament, cardiovascular, intraocular, intraperitoneal, etc. prostheses or implants, or alternatively bioencapsulation systems (implants, microspheres or microcapsules) allowing the sustained and controlled release of active principles in vivo. Medical accessories such as suture threads and also biocompatibilizing coatings for implantable medical articles are other illustrations of the possible uses of the novel biomaterials according to the invention.
For the purposes of the present invention, the term “collagenic peptide” in particular denotes collagen with or without telopeptides, denatured collagen and also gelatin.
Various commercial grades of collagen, with or without telopeptides, are found on the market. These commercial collagens may be of human or animal origin. Collagen is a known protein, which is present at all the levels of organization of animal tissues: it is the main protein of the skin and of connective tissue. By nature, it has biochemical and physicochemical characteristics that are relatively well suited for uses as biomaterials. These characteristics are, in particular: good biocompatibility and biodegradability, hemostatic nature, etc.
However, it must be stated that collagen-based implantable medical, surgical or cosmetic articles suffer from certain shortcomings. They have poor mechanical characteristics, which makes them difficult to handle, or even makes them unusable for certain applications. Furthermore, their biodegradation may be too rapid when the implants need to exert palliative and/or curative functions for long periods. To improve the mechanical and biodegradation characteristics of collagen-based implants, it is found to be necessary to modify the collagen chemically, and in particular to crosslink it.
To modify, in particular to crosslink, collagenic peptides, the reactive functions present on the side chains of certain amino acids of collagen are used, namely:                the amine functions of the lysine residues, representing in numerical terms 3% of the amino acids,        the carboxylic acid functions of the aspartic acids and glutamic acids, representing in numerical terms 9% to 12% of the amino acids,        the alcohol functions of the serine, threonine and hydroxyproline residues, representing in numerical terms 14% of the amino acids.        Thus, four major technical types of artificial crosslinking of this collagenic peptide have appeared.                    1. Creation of a network by covalent bonding between the collagen molecules, by irradiation or forced dehydration. This crosslinking is obtained without chemical functionalization of the collagen.            2. Activation of the natural groups of the collagen, to introduce the possibility of self-crosslinking, for example by oxidation (periodate) or by functional activation (activation of the acids with carbodiimides, in the form of azide . . . which react with the amines).            3. Crosslinking with difunctional or polyfunctional bridging chemical agents (aldehydes, dicarboxylic compounds, diamines, diisocyanates, disulfonyl chlorides or difunctionalized polyethylene glycol).            4. Copolymerization by covalent bonding of the collagen with another polymer (polyacrylic, copolyacrylo-nitrile-styrene, polyurethane, polyalcohol or silicone).                        
One crosslinking variant of type 3 by bridging may consist in using difunctional derivatives containing disulfide groups. This variant is the one which is of interest in the context of the invention. Said variant has given rise in the prior art to various technical propositions, which will be presented below.
The article by F. Schade & H. Zahn [Einbau von cystin-brücken in Kollagen, Angew. Chem., 74, 904, 1962], describes the functionalization of collagen using a cystine derivative, by formation of amide bonds between, on the one hand, the free NH2 moieties of the lysine residues of the collagenic chain and, on the other hand, the carboxyl moieties of the cystine derivative, which have been preactivated by esterification with nitrophenol. The reduction of the disulfide bridges of the grafted cystine derivatives gives a thiolized material which is crosslinkable by oxidation. Since only the lysine residues of the collagen are functionalized, the maximum degree of functionalization, which is directly proportional to the level of crosslinking, is not more than 3% in numerical terms.
European patent application EP 0 049 469 discloses the functionalization of soluble collagen extracted from tendons using N-acetyl homocysteine thiolactone. This is also a case of a reaction between the carboxyl moieties of the functionalizing agent and the amine moieties of the lysine residues of the collagen. The maximum content of grafted thiol functions is thus in this case also not more than 3%.
In order to obtain novel thiolated collagenic derivatives and/or to increase the degrees of grafting of thiol functions on collagen and thereafter the level of crosslinking, the Applicant has proposed, in turn, three novel routes for chemical functionalization of collagen with groups bearing thiol functions or precursors thereof.
The first route is described in French patent FR 2 692 582 which concerns a collagen grafted with thiolated derivatives (cysteine, homocysteine or cysteamine):                via a succinic rotule, one of the carboxyl ends of which has reacted with amine moieties of the lysine residues and with certain alcohol moieties of the serine, threonine and hydroxyproline residues of the collagen and the other carboxyl end of which has reacted with the amine moiety of the thiolated derivative; and        optionally directly without a rotule on the carboxyl functions of the aspartic acids and glutamic acids of the collagen.        
Up to 29% functionalization of the amino acids of the collagen may thus be achieved.
The mercaptoamino functions—that is to say the thiolated derivatives—described in said French patent are attached directly or indirectly to the free NH2, OH and COOH functions of the collagen. Said patent does not disclose a collagenic peptide whose OH and NH2 moieties are functionalized with functions other than mercaptoamino functions.
The second route is given in patent FR 2 699 184 which relates to a collagen grafted with thiolated derivatives (cysteine or homocysteine) attached directly to the amine moieties of the lysine residues and certain alcohol moieties of the serine, threonine and hydroxyproline residues. In accordance with the invention described by said patent, the functionalizing agent (e.g. cystine) which is the precursor of the thiolated derivative grafted onto the collagen comprises an activated carboxyl function, which reacts with the NH2 functions of the lysines to form amides and with the OH functions of the serines, threonines and hydroxyprolines to form esters. This functionalizing agent also comprises a protected amine function, which cannot react with the carboxyls of the aspartic acids and glutamic acids of the collagenic chain. The maximum degree of grafting which may be achieved by this method is 17%.
A third route for the chemical modification of collagen which was developed by the Applicant to provide such a polymer with crosslinking functionality, is described in French patent FR 2 723 957. Said patent discloses a collagen grafted on the free amine moieties of its lysine residues with a thiolated derivative consisting of cysteine or homocysteine whose amine and thiol functions are protected with one and the same protecting group, the whole forming a thiazolidine moiety. The carboxylic acid of the thiazolidine derivative is activated to be able to react with the amine functions of the lysine residues. Consequently, the degree of grafting in this case is not more than 3%. The free carboxylic functions of the glutamic acids and aspartic acids of the collagenic chain are not substituted in the collagen according to said patent.
The collagens according to these three French patents allow the preparation of medical articles (gels, felts, films, etc.) with advantageous levels of crosslinking, that is to say advantageous mechanical and biodegradation characteristics. However, there is scope for their improvement.
Collagens substituted with groups which are not crosslinking functions and which are intended to give the collagen other properties, for example by modifying its solubility characteristics and/or its rheological characteristics and/or its biological characteristics, are moreover known. Thus, patent application PCT WO 90/05755 describes a collagen in which the amines of the lysine residues it comprises are substituted with a synthetic hydrophilic polymer chain and more particularly with monomethyl polyethylene glycol. This collagen-PEG is presented as having low immunogenicity and improved mechanical properties of elasticity and malleability.
Patent application PCT WO 94/01483 discloses a biologically inert, biocompatible conjugated polymer material, formed by a natural polymer such as collagen, linked via an ether bond to a synthetic hydrophilic polymer such as polyethylene glycol (PEG).
The modified collagens according to the prior art do not afford all the desired satisfaction, as regards their mechanical properties, their in vivo degradation kinetics and their biological characteristics. Moreover, the known collagens modified with free or substituted thiol functions still have scope for improvement, as regards controlling, by means of the degree of crosslinking, their mechanical and biological characteristics.
Finally, it would be advantageous for the crosslinkable forms of the known modified collagens to have solubility properties over a wide pH range, so as to make them easier to use, without this having a negative effect on their level of crosslinking.
In this prior art, one of the essential objectives of the invention is to provide novel collagens modified by grafting free or substituted thiol functions, these novel collagens needing to be capable of crosslinking in a sufficient and controlled manner, by forming intercatenary disulfide bridges.
Another essential objective of the invention is to provide novel collagens modified by grafting thiol functions and characterized by high degrees of grafting coexisting with good solubility over a wide pH range.
Another essential objective of the invention is to provide novel collagens modified by grafting thiol functions, that are easy to use and to handle industrially.
Another essential objective of the invention is to provide novel collagens modified by grafting thiol functions, in which the reactive functions are not all mobilized by crosslinking, so as to allow the grafting of noncrosslinking functionalities.
Another essential objective of the invention is to provide novel crosslinkable collagens or crosslinkable collagen precursors that are mercapto-functionalized and able to be converted into gels, films or felts (e.g.) whose crosslinking density (and thus mechanical strength and biodegradation) may be modified beforehand, so as to provide a varied range of starting materials which may be used in numerous applications as biomaterials.
Another essential objective of the invention is to provide a simple process for preparing a collagenic peptide modified by grafting free or substituted thiol functions borne by mercaptoamino residues.